Method of using chemical tags to improve the identification, quantification and spatial localization of components in a sample

ABSTRACT

The present disclosure relates to a method for using chemical tags which have two or more sites for ionization to improve quantification and identification of components of interest from a complex mixture. This method relies on first selectively reacting one or more component in a sample with a chemical tag having two or more sites for ionization, followed by separation of components based on charge status, and finally characterization of each component to identify the same. Additionally disclosed are compounds useful as chemical tags in the disclosed methods.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/297,070, filed Feb. 18, 2016, and U.S. Provisional Application No.62/359,390, filed Jul. 7, 2016, the entire contents of each of which areincorporated herein by reference.

FIELD

This invention relates generally to a method of using an ionizationstate of certain molecules in a complex mixture to isolate, andpotentially quantify, individual components therein. In particular, thepresent disclosure relates to use of chemical tags that contain two ormore potential sites for ionization which selectively react with certaincomponents of a complex mixture. Components of the sample are thenseparated by charge state, with the multiply-charged, tagged componentsbeing easily separated from the other components, followed bycharacterization of all components therein.

BACKGROUND

Biological samples may contain many different types of compounds,including naturally occurring components such as lipids and proteins, aswell as unnaturally occurring components such as small moleculecompounds (e.g., medications), which may be present in the sample.Analysis of biological samples can lead to diagnosis of disease,determination of exposure to, and efficacy of, medications, and thepresence or absence of components of particular interest, among manyother uses.

One means of identifying components in a sample is by using chemicalmodifiers, also known as probes or labels, which have shown utility inthe biology and biotechnology realms as a means to label specificmolecules such as proteins, antibodies or amino acids. There arecurrently several labeling methods for tracking biomolecules such aselectrochemical, optical (including fluorophores), mass change andcalorimetric chemical tags. However, while these chemical modifiers mayhelp in the visualization of a particular component in a solution, theycannot be used to separate components out of a complex sample andcharacterize them.

Another means to determine the components of a biological sample is tosubject it to more traditional analytical methods, such as massspectrometry analysis of the complex mixture of materials in abiological sample. An example of this is shotgun lipidomics. However,such methods often fail to deliver accurate results because of thechemical complexity of the components of samples, i.e., multiple lipidspecies. Thus, this type of approach is often associated with theinability to distinguish isobaric species present in a complex sample.Thus, this method also cannot adequately distinguish and quantify allrelevant species from biological samples.

Currently, there is a need for improved techniques for rapid separationand identification of components from complex mixtures, such asbiological samples.

SUMMARY OF THE INVENTION

The present disclosure relates to a method for using chemical tags whichhave two or more sites for ionization to improve quantification,identification and/or spatial localization of components of interestfrom a complex mixture. This method relies on selectively reacting oneor more component in a sample with a chemical tag having two or moresites for ionization, followed by separation of components based oncharge status, and finally characterization of each component toascertain its identity.

In particular, the present disclosure relates to a method of using theionization state of certain molecules in a complex sample to isolate,and potentially quantify, individual components therein. Methodsdisclosed herein are useful for the identification, quantificationand/or spatial localization of the components of a sample containing oneor more components which may be reacted with a chemical tag containingtwo or more sites of possible ionization.

In one embodiment, the present disclosure relates to a method ofidentifying components of a sample comprising 1) selectively reactingone or more components in the sample with a chemical tag which has twoor more sites for ionization; 2) separating the components using ionmobility spectrometry or capillary electrophoresis; and 3) analyzing thesample to identify the components therein.

In one embodiment, the present disclosure relates to use of a chemicaltag according to one of Formulae (I), (II) or (III):

or a salt thereof, wherein:

each Y is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(k)—,—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—,

each Y′ is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(1c),—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—,

each Y″ is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(k)—, or—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—;

each Y₁ is independently: —(CH₂)_(n)W(CH₂)_(n)—, —OC(O)—(CH₂)₁₋₄—,—OC(O)—(CH₂)₁₋₄W— or absent;

each X is independently: —N⁺(C₁-C₆alkyl)₃, —S(O)₂(OH), —OS(O)₂(OH),—NH—C(═NH)NH₂, —OP(O)(OH)₂, —P(O)(OH)₂, —C(O)N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)₂, —NHC(O)(OC₁-C₆alkyl), —CO₂H, —C(O)NH₂,—C(O)NH(C₁-C₆alkyl), or C(O)(OC₁-C₆alkyl);

each RG is independently isocyanate, thioisocyanate, succinimidyl ester,succinimidyl carbamate, carboxylic acid, amine, or aldehyde;

each W is independently N(C₁-C₆ alkyl) NH, O, S, or absent;

each Z is independently CH or N;

each R is independently halo, —OH, —CN, C₁-C₃ alkyl, C₃-C₆ cycloalkyl,C₁-C₃ alkoxy, C₁-C₃ haloalkyl or C₁-C₃ haloalkoxy;

each n is independently 0-4;

each p is independently 0-6;

each k is independently 0-6; and

m is 0-3;

and wherein each (CH₂) or (CH₂CH₂) segment of Y, Y′ and Y″ may beoptionally and independently substituted with 1 or 2 groups selectedfrom methyl, ethyl, —OH, halo or carbonyl and wherein each Y, Y′ and Y″is attached to an X group; provided that n and k are not both 0 when Y,Y′ or Y″ is —(CH₂W)_(n)(CH₂)_(k)— or —(CH₂CH₂W)_(n)(CH₂)_(k)—.

In a further embodiment, the present disclosure relates to use of achemical tag which is according to one of formulae (Ia), (Ib), (IIa),(IIb), (IIIa), or (IIIb):

or a salt thereof, wherein the variables are as described for formula(I), (II) and (III), above.

In another embodiment, the present disclosure relates to use of achemical tag which is according to one of Formulae (IV), (V), (VI),(VII), (VIII) or (IX):

or a salt thereof; wherein

each Q is N(C₁-C₃alkyl), NH or O;

each T is H, methyl or OH;

each Y₁ is —(CH₂)₁₋₃NH—, —OC(O)(CH₂)₁₋₃—, —NHC(O)(CH₂)₁₋₃—,—C(O)O(CH₂)₁₋₃—, —C(O)NH(CH₂)₁₋₃—, —(CH₂)₁₋₃—, or absent;

each X is independently —N⁺(C₁-C₆alkyl)₃, —S(O)₂(OH), —OS(O)₂(OH),—NHC(═NH)NH₂, —OP(O)(OH)₂, P(O)(OH)₂, —N(C₁-C₆alkyl)₂, or —CO₂H; and

each RG is isocyanate, thioisocyanate, succinimidyl ester or asuccinimidyl carbamate.

Additionally, in accordance with another aspect, the disclosure relatesto novel compounds which can be used as chemical tags in the disclosedmethods. In one embodiment is a compound selected from the groupconsisting of:

or a salt thereof; wherein each R³ is independently C₁-C₆ alkyl; andeach R⁴ is independently —H or C₁-C₆ alkyl.

The methods of the present disclosure provide several advantages overthe prior art. The present methodology may be used for determination ofthe components of a sample with improved selectivity, sensitivity,specificity and/or mass accuracy over present techniques.

Separation of multiply charged species increases resolution of thecomponents and can allow for more efficient separation of targetcomponents from species of same molecular weight (isobaric) as well asfrom impurities. Additional advantages may include the ability toidentify and characterize components such as small molecules, peptidesand/or other biologically relevant molecules from a sample, as well asthe spatial localization of the same. The present method allows forlimited sample preparation and fractionation of the different molecularcontent of the sample prior to analysis to identify the componentstherein. In this way, a multi-omics analysis can be conducted in asingle acquisition. Additionally, the present methods may lead toshorter time to analyze complex samples, by allowing direct infusion ofsamples to be analyzed with minimal to no purification. Shorter time toanalysis is the result of the need for only limited sample preparation,which allows for direct infusion into the MS.

Furthermore, compounds of the present disclosure can be used foranalysis and separation based on charge state for any analyte which isable to react with the chemical tags described herein. Use of chemicaltags of the present invention to selectively tag specific compounds in acomplex sample potentially increases the separation of isobaric species,improves the signal to noise ratio, and thereby reduces false positiveassignments, which in turn increases the accuracy of the results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows analysis of brain extract using an IM-MS instrument; triplycharged and doubly charged ions are highlighted on the figure.

FIG. 2 shows real time analysis of dried blood spots using DESIionization, separation by ion mobility and MS characterization.

DETAILED DESCRIPTION

This invention relates generally to a method of using chemical tags tomodify certain components of a complex sample to isolate, potentiallyquantify, and/or spatially locate, individual components therein basedon charge state, followed by characterization of the individualcomponents. In particular, the method disclosed herein allows forselective reaction of certain components of a complex biological samplewith a chemical tag containing two or more sites for ionization;separation of the components based on charge state using ion mobility(IM) or capillary electrophoresis (CE); and analysis of the sample toidentify the components therein. The methods of the present disclosurecan allow rapid analysis of biological samples without requiring much,if any, sample preparation.

The methods of the present disclosure provide several advantages overthe prior art. The present methodology may be used for the determinationof the components of a sample with improved selectivity, sensitivity,specificity and/or mass accuracy over present techniques.

Definitions

As used herein, the term “isobaric species” refers to a chemicalcompound or molecule that have the same molecular weight. Isobaricspecies can be made up of different atoms, or the same atoms connectedin a different way.

“Dendrimer-type molecule” means a repetitively branched molecule with asingle chemically addressable group (i.e., focal point) which may beuseful to attaching to a molecule in need of chemical tagging.“Dendrimer-type molecules” as useful in the present invention possesstwo or more chemical sites which can be ionized and include moleculestypically characterized as “dendrimers” and “dendrons”, but do notinclude high molecular weight species such as dendrimer-polymers,hyper-branched polymers and/or polymer brushes. Typically, the terms“dendrimers” and “dendrons” include generation 1, generation 2, orgeneration 3 molecules, and/or contain no more than 27 ionizable groups.Generation number refers to the “number of repeated branching cyclesthat are performed during synthesis”

The term “alkyl” means saturated or unsaturated aliphaticstraight-chain, branched or cyclic monovalent hydrocarbon radical.Unless otherwise specified, an alkyl group typically has 1 to 6 carbonatoms, i.e., C₁-C₆-alkyl. As used herein, a “C₁-C₆-alkyl” group means aradical having from 1 to 6 carbon atoms in a linear, branched or cyclicarrangement, and includes methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, isopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethynyl, ethenyl, propynyl, propenyl,and the like.

The term “reactive group” (“RG”) also referred to herein as a“functional group”. Both terms are used interchangeably throughout thespecification. A RG or functional group allows for the reaction betweena component of a sample and a chemical tag according to one of Formulae(I) through (VII). In one embodiment, the RG is a labile group whichallows it to react with a nucleophile or nucleophilic group.Alternatively, the RG is a group which enhances the nucleophilicity of agroup on the chemical tag, such as an amine, which allows it to reactwith an electrophilic group of a component in the sample. In someembodiments, the RG group is able to rapidly react with the targetmolecule or target molecules in the sample. Non-limiting examples of RGgroups include, isocyanates, isothiocyanates, succinimidyl esters,succinimidyl carbamates, carboxylic acids, amines, aldehydes, esters,dienes, alkenes and alkynes. Preferred RG groups include, but are notlimited to, isocyanates, isothiocyanates, succinimidyl esters,succinimidyl carbamates, carboxylic acids, amines, and aldehydes. Asused herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”).

The term “ion-mobility-separation” (“IM”) is a gas-phase electrophoretictechnique that enables the separation of gas-phase lipid ions within achamber pressurized with a buffer gas, such as purified argon ornitrogen gas. An inert gas is not necessarily elemental and is often acompound gas that have the tendency for non-reactivity is due to thevalence, the outermost electron shell, being complete in all the inertgases. This is a tendency, not a rule, as noble gases and other “inert”gases can react to form compounds.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

“Spectroscopy” refers to the study of the interaction between matter andelectromagnetic radiation. Spectroscopic data is often represented by anemission spectrum, a plot of the response of interest as a function ofwavelength or frequency. Exemplary spectroscopic techniques includeMossbauer spectroscopy. X-ray absorption spectroscopy, UV/Visspectroscopy, atomic absorption spectroscopy, infrared spectroscopy,Raman spectroscopy, microwave spectroscopy, electron spin resonancespectroscopy, nuclear magnetic resonance spectroscopy (NMR), nuclearquadrupole resonance spectroscopy (NQR), nuclear atomic emissionspectroscopy, X-ray fluorescence, fluorescence spectroscopy,phosphorescence spectroscopy, atomic fluorescence spectroscopy, andchemiluminescence spectroscopy, each of which can provide uniqueinformation about the properties of a chemical species. For example,Raman spectroscopy is a spectroscopic technique used to identifymolecules by their characteristic vibrational, rotational and otherlow-frequency modes.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include (but are not limited to) reverse phase liquidchromatography (RPLC), high performance liquid chromatography (HPLC),ultra-high performance liquid chromatography (UPLC or UHPLC), turbulentflow liquid chromatography (TFLC) (sometimes known as high turbulenceliquid chromatography (HTLC) or high throughput liquid chromatography),and carbon dioxide based chromatograpy.

As used herein, the term “high performance liquid chromatography” or“HPLC” (also sometimes known as “high pressure liquid chromatography”)refers to liquid chromatography in which the degree of separation isincreased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column. As used herein, theterm “ultra high performance liquid chromatography” or “UHPLC”(sometimes known as “ultra high pressure liquid chromatography”) refersto HPLC that occurs at much higher pressures than traditional HPLCtechniques.

The term “LC/MS” refers to a liquid chromatograph (LC) interfaced to amass spectrometer. The term “LC/MS/MS” refers to a liquid chromatograph(LC) interfaced to an instrument that includes two mass spectrometers.

The term “IM-MS” refers to method that separates gas phase ions on amillisecond timescale using ion-mobility spectrometry and uses massspectrometry on a microsecond timescale to identify components in asample.

The term “drift time” refers to the time required for lipid ions tocross the ion-mobility separation cell. This net ion motion is usuallymuch slower than the normally occurring random motion. In asemiconductor the charge carriers will typically have different driftvelocities for the same electric field. SI unit of mobility is(m/s)/(V/m)=m²/(V·s). However, mobility is much more commonly expressedin cm²/(V·s)=10⁻⁴ m²/(V·s).

The term “collision cross section” (“CSS”) refers to an area thatquantifies the likelihood of a scattering event when an incident speciesstrikes a target species. In a hard object approximation, the crosssection is the area of the conventional geometric cross section. Thecollisional cross sections typically denoted a and measured in units ofarea.

Methods of the Invention

The methods of the present disclosure provide several advantages overthe prior art. The present methodology may be used for the determinationof the components of a sample with improved selectivity, sensitivity,specificity and/or mass accuracy over present techniques. Separation ofmultiply charged species increases resolution of components in amixture, and can allow for more efficient separation of targetcomponents from species of similar weight, as well as from impurities.Additional advantages include the ability to identify and characterizecomponents such as small molecules, peptides and/or other biologicallyrelevant molecules from a sample, and the spatial localization of thesame. The present method may allow for limited sample preparation andfractionation of the different molecular content of the sample prior toanalysis to identify the components there. In this way, a multi-omicsanalysis may be conducted in a single acquisition. Additionally, thepresent methods may lead to shorter time to analyze complex samples, byallowing direct infusion of samples to be analyzed with minimal to nopurification. In one embodiment, the present disclosure relates to amethod of identifying components of a sample comprising 1) selectivelyreacting one or more components in the sample with a chemical tag whichhas two or more sites for ionization; 2) separating the components usingion mobility spectrometry or capillary electrophoresis; and 3) analyzingthe sample to identify the components therein.

In another embodiment, the present method includes the additional stepof separating the molecules in a biological sample using chromatographyafter the reaction of specific components with a chemical tag. Thischromatographic step may then be followed by separation of the ionizedmolecules of the sample according to the ionization state of thecomponents and analysis of the sample to identify the componentstherein. In a further embodiment, the present method includes the stepsof: 1) selectively reacting a component in the sample with a chemicaltag which has two or more sites for ionization; 2) separation of thetagged biological sample by chromatography; 3) ionizing the componentsof the sample; 4) separating the ionized components of the sample usingion mobility spectrometry or capillary electrophoresis; and 5) analyzingthe sample to identify the components therein.

In either of the above two embodiments, the separation of the biologicalsample by chromatography includes separation by gas chromatography orliquid chromatography. In one aspect of this embodiment, the gaschromatography may be carbon-dioxide based supercritical fluidchromatography. In one aspect of this embodiment, the separation of thebiological sample is done using liquid chromatography, wherein theliquid chromatography is selected from ultra-high performance liquidchromatography (UHPLC), traditional low-pres sure liquid chromatography,high performance liquid chromatography (HPLC), and hydrophilicinteraction chromatography (HILIC). In one aspect of this embodiment,HPLC may refer to normal phase chromatography or reverse phasechromatography (RPC). The resulting method may provide an additionaldegree of separation and will result increased specificity of componentsto selectively react with the desired chemical tag, and thus, ultimatelyenhance identification and relative quantification.

In another embodiment, the present method includes use of any type ofmass spectrometer to analyze the sample and identify the componentstherein (after the separation by ion mobility or capillaryelectrophoresis) to identify and possibly quantify the individualcomponents of the sample. In one aspect of this embodiment, the presentmethod includes analysis of the separated ionized molecules after ionmobility separation using sector, time-of-flight, quadrupole, ion trap,or Fourier transform ion cyclotron resonance, or by tandem massspectrometry (MS/MS) (where two or more of the above types are combinedin tandem or orthogonally). In one embodiment, the present disclosureincludes analysis of the separated ionized molecules after ion mobilityseparation is done using tandem mass spectrometry (MS/MS).

In yet another embodiment, the present disclosure includes analyzing asample, comprising selectively reacting one or more component in thesample with a chemical tag which has two or more sites for ionizationand an additional functionality, such as a fluorescent moiety or amoiety which is visible in the IR or UV-Vis spectrum, separatingcomponents using ion mobility spectrometry or capillary electrophoresis,and analyzing the sample at a plurality of different locations andidentifying the components contained in location sampled. In thisembodiment, the spatial localization will be enhanced by the additionalproperties of the chemical tag, which would allow for visualization oftagged components without the need for MS or additional analyticalmethods.

In one embodiment, the method of the present disclosure may be performedby incorporation ion mobility separation into the Waters TechnologiesCorporation MS^(E) process (for example, processes capable with usingXevo® GS-XS QTof, SYNAPT® G2-Si MS, Vion® IMS QTof, all commerciallyavailable from Waters Technologies Corporation, Milford, Mass.). Use ofthis process for the present method allows an acquisition mode, highdefinition MS (HDMSE), where co-eluting lipid precursor ions can beseparated by ion-mobility before fragmentation, resulting in cleanerMS/MS product-ion spectra. In aspect of this embodiment, the methodincludes the calculation of the CCS value for the ionized molecules. Ina further aspect of this embodiment, the CCS value calculated in thepresent method is used to assist in the identification of components ofthe sample. The methods, processes, and techniques in accordance to thepresent disclosure provide a technological advancement with respect tothe information obtained in comparison to the prior art.

In another embodiment, the present disclosure relates to separation ofisobaric components from a biological sample based on the charge stateof the ionized molecules by first separating the ionized componentsaccording to the IM or CE of the ionized components, and then analyzingthe ionized components using an analytical technique such as massspectrometry. In one aspect of this embodiment, the biological sampleincludes one or more isobaric components, one of which is selectivelyreacted with a chemical tag of the invention, which can be separatedusing differential charge status meaning one isobaric species can bereacted with a chemical tag having two or more possible sites forionization, while the other isobaric species has a different number ofpossible sites for ionization, such as one, and the ion separation stepis able to separate out the isobaric species based on charge status.

Advantages of the present method include the ability to obtainreal-time, high-throughput analysis of biological samples such as driedblood spots, biofluids and tissue-biopsies without performing much, ifany, sample preparation. The presently disclosed methodology can be usedfor determination of individual components from a sample with improvedselectivity, sensitivity, specificity, and/or mass accuracy over currentseparation techniques, while maintaining the ability to rapidly analyzesamples.

Compounds of the Invention

Compounds of the present disclosure can be used for analysis andseparation based on charge state for any analyte that is able to reactwith the chemical tags described herein.

In one embodiment, the present disclosure relates to use of a chemicaltag according to one of Formulae (I), (II) or (III):

or a salt thereof, wherein:

each Y is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(k)—,—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—,

each Y′ is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)C_(k)—,—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—,

each Y″ is independently —(CH₂CH₂)X—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(k)—, or—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—;

each Y₁ is independently: —(CH₂)_(n)W(CH₂)_(n)—, —OC(O)—(CH₂)₁₋₄—,—OC(O)—(CH₂)₁₋₄W— or absent;

each X is independently: —N⁺(C₁-C₆alkyl)₃, —S(O)₂(OH), —OS(O)₂(OH),—NH—C(═NH)NH₂, —OP(O)(OH)₂, —P(O)(OH)₂, —C(O)N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)₂, —NHC(O)(OC₁-C₆alkyl), —CO₂H, —C(O)NH₂,—C(O)NH(C₁-C₆alkyl), or C(O)(OC₁-C₆alkyl);

each RG is independently isocyanate, thioisocyanate, succinimidyl ester,succinimidyl carbamate, carboxylic acid, amine, or aldehyde;

each W is independently N(C₁-C₆ alkyl) NH, O, S, or absent;

each Z is independently CH or N;

each R is independently halo, —OH, —CN, C₁-C₃ alkyl, C₃-C₆ cycloalkyl,C₁-C₃ alkoxy, C₁-C₃ haloalkyl or C₁-C₃ haloalkoxy;

each n is independently 0-4;

each p is independently 0-6;

each k is independently 0-6; and

m is 0-3;

and wherein each (CH₂) or (CH₂CH₂) segment of Y, Y′ and Y″ may beoptionally and independently substituted with 1 or 2 groups selectedfrom methyl, ethyl, —OH, halo or carbonyl and wherein each Y, Y′ and Y″is attached to an X group; provided that n and k are not both 0 when Y,Y′ or Y″ is —(CH₂W)_(n)(CH₂)_(k)— or —(CH₂CH₂W)_(n)(CH₂)_(k)—.

Chemical Tags

Chemical tags are used in present invention to selectively tag specificcompounds in a complex sample potentially increases separation fromisobaric species, improving the signal to noise ratio, and therebyreduces false positive assignments, which increases the measurementaccuracy of the results.

In one embodiment, the present disclosure relates to use of a chemicaltag which has two or more sites of possible ionization. In one aspect ofthis embodiment, the chemical tag may also contain a fluorescent orultra-violet (UV) active moiety. In another aspect of this embodiment,the chemical tag may contain a moiety which absorbs in the visible (VIS)spectrum or the infrared (IR) spectrum.

In another embodiment, the present disclosure relates to use of achemical tag which has two or more site of possible ionization. In oneaspect of this embodiment, the groups which have the potential forionization include, but are not limited to, amine, trialkyl ammonium,guanidine, carboxylic acid, ester (ideally after hydrolysis), amide(ideally after hydrolysis), sulfate, sulfonate, phosphate and/orphosphonate. In one aspect of this embodiment, the ionizable group is acarboxylic acid. In another aspect of this embodiment, the ionizablegroup is an amine. In another aspect of this embodiment, the ionizablegroup is a phosphonate. In another aspect of this embodiment, theionizable group is a phosphate. In another aspect of this embodiment,the ionizable group is a sulfonate. In another aspect of thisembodiment, the ionizable group is a guanidinyl. In another aspect ofthis embodiment, the ionizable group is a trialkyl ammonium group. Inanother aspect of this embodiment, the ionizable group is a sulfate. Ina further aspect of this embodiment, the ionizable group is an ester,which may be hydrolyzed to its corresponding carboxylic acid. In afurther aspect of this embodiment, the ionizable group is an amide,which may be hydrolyzed to its corresponding carboxylic acid.

In yet another embodiment, the present disclosure relates to use of achemical tag which is according to one of formulae (Ia), (Ib), (IIa),(IIb), (IIIa), or (IIIb):

or a salt thereof, wherein the variables are as described for formula(I), (II) and (III), above.

In one aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each Y, Y′ andY″ is independently selected from [(CH₂)₁₋₂OC(O)(OH₂)₁₋₂]₁₋₂—,—[(CH₂)₁₋₂C(O)O(CH₂)₁₋₂]₁₋₂—, —[(CH₂)₁₋₂O(CH₂)₁₋₃]₁₋₂—,—[(CH₂)₁₋₂C(O)NH(CH₂)₁₋₂]₁₋₂— and —[(CH₂)₁₋₂NHC(O)(CH₂)₁₋₂]₁₋₂—, whereinone or two hydrogens of each CH₂ group may be optionally andindependently replaced with a group selected from CH₃ and —OH; each Y₁is —(CH₂)₁₋₃NH—, —OC(O)(CH₂)₁₋₃—, —NHC(O)(CH₂)₁₋₃—, —C(O)O(CH₂)₁₋₃—,—C(O)NH(CH₂)₁₋₃—, —(CH₂)₁₋₃—, or absent; each X is independently—N⁺(C₁-C₆alkyl)₃, —S(O)₂(OH), —OS(O)₂(OH), —NHC(═NH)NH₂, —OP(O)(OH)₂,P(O)(OH)₂, —N(C₁-C₆alkyl)₂, or —CO₂H; and each RG is isocyanate,thioisocyanate, succinimidyl ester or a succinimidyl carbamate.

In another aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each n is aninteger from 0 to 4. In one aspect of this embodiment, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), nis 0. In one aspect of this embodiment, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), n is 1. In oneaspect of this embodiment, for any one of Formulae (I), (Ia), (Ib),(II), (IIa), (IIb), (III), (IIIa) or (IIIb), n is 2. In one aspect ofthis embodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), n is 3. In one aspect of thisembodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb),(III), (IIIa) or (IIIb), n is 4.

In yet another aspect of the above embodiments, for any one of Formulae(I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each p isan integer from 0 to 6. In one aspect of this embodiment, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), pis 0. In one aspect of this embodiment, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), p is 1. In oneaspect of this embodiment, for any one of Formulae (I), (Ia), (Ib),(II), (IIa), (IIb), (III), (IIIa) or (IIIb), p is 2. In one aspect ofthis embodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), p is 3. In one aspect of thisembodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb),(III), (IIIa) or (IIIb), p is 4. In one aspect of this embodiment, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), p is 5. In one aspect of this embodiment, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), pis 6.

In still another aspect of the above embodiments, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb),each k is an integer from 0 to 6. In another aspect of this embodiment,for any one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III),(IIIa) or (IIIb), k is 0. In one aspect of this embodiment, for any oneof Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), k is 1. In one aspect of this embodiment, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), kis 2. In one aspect of this embodiment, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), k is 3. In oneaspect of this embodiment, for any one of Formulae (I), (Ia), (Ib),(II), (IIa), (IIb), (III), (IIIa) or (IIIb), k is 4. In one aspect ofthis embodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), k is 5. In one aspect of thisembodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb),(III), (IIIa) or (IIIb), k is 6.

In another aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each m is aninteger from 0 to 3. In one aspect of this embodiment, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), mis 0. In one aspect of this embodiment, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), m is 1. In oneaspect of this embodiment, for any one of Formulae (I), (Ia), (Ib),(II), (IIa), (IIb), (III), (IIIa) or (IIIb), m is 2. In one aspect ofthis embodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), m is 3.

In yet another aspect of the above embodiments, for any one of Formulae(III), (IIIa) or (IIIb), each R is independently halo, —OH, —CN, C₁-C₃alkyl, C₃-C₆ cycloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkyl or C₁-C₃haloalkoxy. In a further aspect of the above embodiments, for any one ofFormulae (III), (IIIa) or (IIIb), R is independently —F or —Cl. In afurther aspect of the above embodiments, for any one of Formulae (III),(IIIa) or (IIIb), R is independently methyl. In a further aspect of theabove embodiments, for any one of Formulae (III), (IIIa) or (IIIb), R isindependently methoxy. In a further aspect of the above embodiments, forany one of Formulae (III), (IIIa) or (IIIb), R is independently CF₃. Ina further aspect of the above embodiments, for any one of Formulae(III), (IIIa) or (IIIb), R is independently OCF₃. In a further aspect ofthe above embodiments, for any one of Formulae (III), (IIIa) or (IIIb),R is independently cyclopropyl.

In still another aspect of the above embodiments, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb),Y₁ is independently —(CH₂)_(n)W(CH₂)_(n)—, —OC(O)—(CH₂)₁₋₄—,—OC(O)—(CH₂)₁₋₄W— or absent. In one aspect of the above embodiments, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), Y₁ is absent. In one aspect of the above embodiments, for anyone of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y₁ is —OC(O)—(CH₂)₁₋₄—. In one aspect of the above embodiments,for any one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III),(IIIa) or (IIIb), Y₁ is —OC(O)—(CH₂)₁₋₄W—.

In another aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each W isindependently N(C₁-C₆ alkyl), NH, S or O. In one aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), each W is independently N(C₁-C₆ alkyl).In one aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each W isindependently NH. In one aspect of the above embodiments, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb),each W is independently O. In one aspect of the above embodiments, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), each W is independently S.

In yet another aspect of the above embodiments, for any one of Formulae(I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each Y, Y′and Y″ is independently selected from —[(CH₂)OC(O)(CH₂)₂]₂—,—[(CH₂)₂OC(O)(CH₂)]₂—, —(CH₂)OC(O)(CH₂)₂—, —(CH₂)₂OC(O)(CH₂)—,—[(CH₂)C(O)O(CH₂)₂]₂—, [(CH₂)₂C(O)O(CH₂)]₂—, —(CH₂)C(O)O(CH₂)₂—,—(CH₂)₂C(O)O(CH₂)—, —[(CH₂)O(CH₂)₃]₂—, —(CH₂)O(CH₂)₃—,—[(CH₂)₂O(CH₂)₃]₂—, —(CH₂)₂O(CH₂)₃—, —[(CH₂)₂O(CH₂)₂]₂—,—(CH₂)₂O(CH₂)₂—. —[(CH₂)NHC(O)(CH₂)₂]₂—, —[(CH₂)₂NHC(O)(CH₂)]₂—,—(CH₂)NHC(O)(CH₂)₂—, —(CH₂)₂NHC(O)(CH₂)—, —[(CH₂)C(O)NH(CH₂)₂]₂—,—[(CH₂)₂C(O)NH(CH₂)]₂—, —(CH₂)C(O)NH(CH₂)₂—, and —(CH₂)₂C(O)NH(CH₂)—,and wherein one or two hydrogens of each CH₂ group may be optionally andindependently replaced with a group selected from —CH₃ and —OH.

In still another aspect of the above embodiments, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (I b), (III), (IIa) or (IIIb), Y,Y′ and Y″ are independently absent. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIa) or (IIIb), Y, Y′ and Y″ are independently —NH—. Inanother aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″are independently a C₁-C₆ alkyl. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independentlyN(C₁-C₆alkyl)-. In another aspect of the above embodiments, for any oneof Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y, Y′ and Y″ are independently —[(CH₂)OC(O)(CH₂)₂]₂—. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ areindependently —[(CH₂)₂OC(O)(CH₂)]₂—. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—(CH₂)OC(O)(CH₂)₂—. In another aspect of the above embodiments, for anyone of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y, Y′ and Y″ are independently —(CH₂)₂OC(O)(CH₂)—. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ areindependently —[(CH₂)C(O)O(CH₂)₂]₂—. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—[(CH₂)₂C(O)O(CH₂)]₂—. In another aspect of the above embodiments, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), Y, Y′ or Y″ is —(CH₂)C(O)O(CH₂)₂—. In another aspect of theabove embodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—(CH₂)₂C(O)O(CH₂)—. In another aspect of the above embodiments, for anyone of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y, Y′ and Y″ are independently —[(CH₂)O(CH₂)₃]₂—. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ areindependently —(CH₂)O(CH₂)₃—. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—[(CH₂)₂O(CH₂)₃]₂—. In another aspect of the above embodiments, for anyone of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y, Y′ and Y″ are independently —(CH₂)₂O(CH₂)₃—. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ areindependently —[(CH₂)₂O(CH₂)₂]₂—. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—(CH₂)₂O(CH₂)₂—. In another aspect of the above embodiments, for any oneof Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y, Y′ and Y″ are independently —[(CH₂)NHC(O)(CH₂)₂]₂—. Inanother aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″are independently —[(CH₂)₂NHC(O)(CH₂)]₂—. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—(CH₂)NHC(O)(CH₂)₂—. In another aspect of the above embodiments, for anyone of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), Y, Y′ and Y″ are independently —(CH₂)₂NHC(O)(CH₂)—. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ areindependently —[(CH₂)C(O)NH(CH₂)₂]₂—. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″ are independently—[(CH₂)₂C(O)NH(CH₂)]₂—. In another aspect of the above embodiments, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), Y, Y′ and Y″ are independently —(CH₂)C(O)NH(CH₂)₂—. Inanother aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), Y, Y′ and Y″are independently —(CH₂)₂C(O)NH(CH₂)—.

In yet another aspect of the above embodiments, for any one of Formulae(I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), eachinstance of X are all the same. In aspect of the above embodiments, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), each X is N(C₁-C₆ alkyl)₂. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), UM, (IIIa) or (IIIb), each X is N⁺(C₁-C₆ alkyl)₃. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each X is N(CH₃)₂. Inanother aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each X isN(CH₂CH₃)₂. In another aspect of the above embodiments, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb),each X is —CO₂H. In another aspect of the above embodiments, for any oneof Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), each X is —CO₂CH₃ or —CO₂(CH₂CH₃). In another aspect of theabove embodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), each X is —S(O)(OH)₂. In another aspectof the above embodiments, for any one of Formulae (I), (Ia), (Ib), (II),(IIa), (IIb), (III), (IIIa) or (IIIb), each X is —OS(O)(OH)₂. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each X isNHC(═NH)NH₂. In another aspect of the above embodiments, for any one ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb),each X is OP(O)(OH)₂. In another aspect of the above embodiments, forany one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa)or (IIIb), each X is P(O)(OH)₂. In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), each X is C(O)N(C₁-C₆alkyl)₂. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each X is C(O)NH₂. Inanother aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or (IIIb), each X isC(O)NH(C₁-C₆alkyl). In another aspect of the above embodiments, for anyone of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) or(IIIb), each X is —NHC(O)(C₁-C₆alkyl). In another aspect of the aboveembodiments, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), each X is C(O)(OC₁-C₆alkyl).

In another embodiment, for any one of Formulae (I), (Ia), (Ib), (II),(IIa), (IIb), (III), (IIIa) or (IIIb), RG is a succinimidyl ester group.In one embodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa) or (IIIb), RG is a succinimidyl carbamate group. Inanother embodiment, for any one of Formulae (I), (Ia), (Ib), (II),(IIa), (IIb), (III), (IIIa) or (IIIb), RG is an isocyanate group. Inanother embodiment, for any one of Formulae (I), (Ia), (Ib), (II),(IIa), (IIb), (III), (IIIa) or (IIIb), RG is a thioisocyanate group. Inanother embodiment, for any one of Formulae (I), (Ia), (Ib), (II),(IIa), (IIb), (III), (IIIa) or (IIIb), RG is a carboxylic acid group. Inanother embodiment, for any one of Formulae (I), (Ia), (Ib), (II),(IIa), (IIb), (III), (IIIa) or (IIIb), RG is an amine group. In anotherembodiment, for any one of Formulae (I), (Ia), (Ib), (II), (IIa), (IIb),(III), (IIIa) or (IIIb), RG is an aldehyde group.

In yet another embodiment, the present disclosure relates to use of achemical tag according to one of Formulae (IV), (V), (VI), (VII), (VIII)or (IX):

or a salt thereof; wherein each Q is N(C₁-C₃alkyl), NH or O; each T isH, methyl or OH; each Y₁ is —(CH₂)₁₋₃NH—, —OC(O)(OH₂)₁₋₃—,—NHC(O)(CH₂)₁₋₃—, —C(O)O(CH₂)₁₋₃—, —C(O)NH(CH₂)₁₋₃—, —(CH₂)₁₋₃—, orabsent; each X is independently —N⁺(C₁-C₆alkyl)₃, —S(O)(OH)₂,—OS(O)(OH)₂, —NHC(═NH)NH₂, —OP(O)(OH)₂, —P(O)(OH)₂, —N(C₁-C₆alkyl)₂, or—CO₂H; and each RG is isocyanate, thioisocyanate, succinimidyl ester ora succinimidyl carbamate.

In one aspect of the above embodiments, for any one of Formulae (I),(Ia), (Ib), (III), (IIIa), (IIIb), (VII) or (VIII) Z is N. In anotheraspect of the above embodiments, for any one of Formulae (I), (Ia),(Ib), (III), (IIIa), (IIIb), (VII) or (VIII), Z is CH.

In another aspect of the above embodiments, for Formula (VI), each T is—H. In one aspect of the above embodiments, for Formula (VI), each T is—OH. In one aspect of the above embodiments, for Formula (VI), each T isCH₃.

In yet another aspect of the above embodiments, for any one of Formulae(IV), (V), (VI), (VII), (VIII), or (IX), each instance of X are all thesame. In aspect of the above embodiments, for any one of Formulae (IV),(V), (VI), (VII), (VIII), or (IX), each X is N(C₁-C₆ alkyl)₂. In anotheraspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), each X is N⁺(C₁-C₆ alkyl)₃. In anotheraspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), each X is N(CH₃)₂. In another aspect ofthe above embodiments, for any one of Formulae (IV), (V), (VI), (VII),(VIII), or (IX), each X is —N(CH₂CH₃)₂. In another aspect of the aboveembodiments, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), each X is —CO₂H. In another aspect of the above embodiments, forany one of Formulae (IV), (V), (VI), (VII), (VIII), or (IX), each X isS(O)(OH)₂. In another aspect of the above embodiments, for any one ofFormulae (IV), (V), (VI), (VII), (VIII), or (IX), each X is OS(O)(OH)₂.In another aspect of the above embodiments, for any one of Formulae(IV), (V), (VI), (VII), (VIII), or (IX), each X is NHC(═NH)NH₂. Inanother aspect of the above embodiments, for any one of Formulae (IV),(V), (VI), (VII), (VIII), or (IX), each X is —OP(O)(OH)₂. In anotheraspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), each X is P(O)(OH)₂.

In still another embodiment, for any one of Formulae (IV), (V), (VI),(VII), (VIII), or (IX), RG is a succinimidyl ester group. In oneembodiment, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), RG is a succinimidyl carbamate group. In another embodiment, forany one of Formulae (IV), (V), (VI), (VII), (VIII), or (IX)), RG is anisocyanate group. In another embodiment, for any one of Formulae (IV),(V), (VI), (VII), (VIII), or (IX), RG is a thioisocyanate group.

In another aspect of the above embodiments, for any one of Formulae(IV), (V), (VI), (VII), (VIII), or (IX), each Y₁ is independently—(CH₂)₁₋₃NH—, —OC(O)(CH₂)₁₋₃—, —NHC(O)(CH₂)₁₋₃—, —C(O)O(CH₂)₁₋₃—,—C(O)NH(CH₂)₁₋₃—, —(CH₂)₁₋₃—, or absent. In one aspect of the aboveembodiments, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), Y₁ is absent. In one aspect of the above embodiments, for any oneof Formulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ is CH₂—. In oneaspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), Y₁ is CH₂CH₂—. In one aspect of the aboveembodiments, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), Y₁ is (CH₂)₃—. In one aspect of the above embodiments, for any oneof Formulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ is CH₂NH—. Inone aspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), Y₁ is CH₂CH₂NH—. In one aspect of theabove embodiments, for any one of Formulae (IV), (V), (VI), (VII),(VIII), or (IX), Y₁ is (CH₂)₃NH—. In one aspect of the aboveembodiments, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), Y₁ is OC(O)CH₂—. In one aspect of the above embodiments, for anyone of Formulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ isOC(O)CH₂CH₂—. In one aspect of the above embodiments, for any one ofFormulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ is OC(O)(CH₂)₃—. Inone aspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), Y₁ is —NHC(O)CH₂—. In one aspect of theabove embodiments, for any one of Formulae (IV), (V), (VI), (VII),(VIII), or (IX), Y₁ is NHC(O)CH₂CH₂—. In one aspect of the aboveembodiments, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), Y₁ is NHC(O)(CH₂)₃—. In one aspect of the above embodiments, forany one of Formulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ is—C(O)OCH₂—. In one aspect of the above embodiments, for any one ofFormulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ is C(O)OCH₂CH₂—. Inone aspect of the above embodiments, for any one of Formulae (IV), (V),(VI), (VII), (VIII), or (IX), Y₁ is C(O)O(CH₂)₃—. In one aspect of theabove embodiments, for any one of Formulae (IV), (V), (VI), (VII),(VIII), or (IX), Y₁ is —C(O)NHCH₂—. In one aspect of the aboveembodiments, for any one of Formulae (IV), (V), (VI), (VII), (VIII), or(IX), Y₁ is C(O)NHCH₂CH₂—. In one aspect of the above embodiments, forany one of Formulae (IV), (V), (VI), (VII), (VIII), or (IX), Y₁ isC(O)NH(CH₂)₃—.

The present disclosure includes use of compounds according to any ofFormulae (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb),(IV), (V), (VI), (VII), (VIII) and (IX) as chemical tags in thedisclosed methods, wherein the particular value for any of the variablesY, Y′, Y″, Y1, X, W, RG, Z, R, n, p, k, m, Q, and T, where present, maybe combined with any other particular value for the remaining variables,as disclosed above.

The present disclosure also includes compounds according to any ofFormulae (I), (Ia), (Ib), (II), (Ila), (IIb), (III), (IIIa), (IIIb),(IV), (V), (VI), (VII), (VIII) and (IX).

In one embodiment, wherein the chemical tag is a dendrimer typemolecule, the dendrimer is a polyamindoamine (PAMAM), polypropyleneimine(PPI) a polyglutamic acid and/or a polyester dendrimer.

In another embodiment of the present invention, the chemical tag isselected from the group consisting of:

or a salt thereof.

In a further embodiment, the chemical tag is selected from:

or a salt thereof; wherein each R³ is independently C₁-C₆ alkyl; andeach R⁴ is independently —H or C₁-C₆ alkyl. In one aspect of thisembodiment, each R³ is methyl and each R⁴ is H or methyl. In anotheraspect of this embodiment, each R³ is methyl and each R⁴ is H.

Dendrimer-type compounds of formulae (I), (Ia), (Ib), (II), (IIa),(IIb), (III), (IIIa), (IIIb), (IV), (V), (VI), (VII), (VIII), and (IX)can be made according the methods known in the art. See, e.g., Tomaliaet al., A New Class of Polymers: Starburst-Dendritic Macromolecules,Polymer Journal (1985), 17:117-132; Tomalia et al., Poly(amidoamine)(PAMAM) Dendrimers: from biomimicry to drug delivery and biomedicalapplications, Drug Discovery Today (2001), 15:427-436; Newkome et al.,Micelles, Part I. Cascade Molecules: a new approach to micelles, J. Org.Chem. (1985), 50(11):2003-2004; and Hawker et al., Preparation ofPolymers with Controlled Molecular Architecture, J. Am. Chem. Soc.(1990), 112(21):7638-7647; Klajnert, Barbara and Bryszewska, Maria;Dendrimers: Properties and Applications, Acta Biochimica Polonica,(2001) 48(1):199-208.

Additional compounds of formulae (I), (Ia), (Ib), (II), (IIa), (IIb),(III), (IIIa), (IIIb), (IV), (V), (VI), (VII), (VIII), and (IX) can besynthesized using standard organic methodologies, with use of standardprotection/deprotection techniques as necessary. See Nicolaou, K. C.;Sorensen, E. J. (1996), Classics in Total Synthesis. New York: VCH;March, J.; Smith, D, (2001), Advanced Organic Chemistry, 5th ed. NewYork: Wiley; Clayden, Jonathan; Greeves, Nick; Warren, Stuart; Wothers,Peter, (2000), Organic Chemistry, Oxford University Press.

Separation of Lipid Ions

Ion Mobility (IM) & Capillary Electrophoresis (CE)

IM is a gas-phase electrophoretic technique that enables the separationof gas-phase lipid ions within a chamber pressurized with a buffer gas(e.g., nitrogen). Capillary electrophoresis is an electrokineticseparation method performed in sub-millimeter capillaries and in micro-and nanofluidic channels.

In one embodiment, the present disclosure includes separation ofcomponents of a sample based on charge state using either ion mobility(IM).

During IM, the time required for lipid ions to cross the ion-mobilityseparation cell—the drift time—depends principally on the collisionfrequency between the ions and the buffer gas. Thus drift times aredirectly related to the shape, size, and charge of the component ions aswell as to the nature of the buffer gas. From the characteristic timethat a component ion takes to cross the ion mobility separation cell(drift time), it is possible to calculate the rotationally-averagedcollision cross section (CCS), which represents the effective area forthe interaction between an individual ion and the neutral gas throughwhich it travels. CCS, an important physicochemical property ofcompounds, is related to chemical structure and three-dimensionalconformation. Changing the size of the chemical tag will alter the CCSvalue for any tagged component in a sample. Thus, the present disclosureallows for selective increase of the CCS value for components in asample by using bulkier chemical tags. By controlling the charge and thesize of the chemical tag, it may be possible to significantly affect theseparation of the components of a sample in order to better quantify andcharacterize them. In other words, IM allows separating multiply chargedmolecules by their charge status, pulling them apart from the rest ofthe molecules in the sample, and use of further analysis using CCS datacan improve their identification, quantification and/or spatiallocalization. See FIG. 1 which shows doubly and triply charged lipidions being specifically delineated from a sample containing a multitudeof singly charged ion species.

Components in a sample may localize in different compositions andconcentrations across its surface. Imaging of samples (analyzing usingthe techniques disclosed herein) may allow topographic mapping (i.e.,spatial localization) of distinct components in a sample (e.g., cellcultures and tissue sections). For example, in a typical IM-MS imagingexperiment, a focused excitatory beam (e.g., laser or a stream ofcharged solvent droplets) is directed at the biological sample to scanthe surface along a user defined two dimensional array. Upon impact ofthe excitatory beam, biomolecular ions are desorbed and ionized from thesample surface and directed into the mass spectrometer. The addition ofIM to a typical MS imaging experiment allows separation of thecomponents of interest from the interfering background before MSdetection, resulting in a greater signal-to-noise ratio and moreaccurate localization of the same. In one embodiment, IM is followed byidentification of the components of the sample using UV spectroscopy,flame ion detection, Raman spectroscopy, or nuclear quadrupoleresonance.

In another embodiment, the present disclosure includes analyzing asample, comprising selectively reacting one or more component in thesample with a chemical tag which has two or more sites for ionization,separating components using ion mobility spectrometry or capillaryelectrophoresis, and analyzing the sample at a plurality of differentlocations and identifying the components contained in location sampled.In one aspect of this embodiment, the components are first separatedusing IM, and then analyzed using MS. In another aspect of thisembodiment, the data collected by the method for each location is usedto determine the spatial localization of molecules in the sample. In oneaspect of these embodiments, the biological sample is a tissue sectionor a cell culture.

Capillary electrophoresis (CE) may refer to capillary zoneelectrophoresis (CZE), capillary gel electrophoresis (CGE), capillaryisoelectric focusing (LIEF) and/or capillary isotachophoresis andmicellar electrokinetic chromatography (MEKC). Separation of ionizedspecies occurs due to their electrophoretic mobility. In one embodiment,the present disclosure relates to use of capillary electrophoresis forseparation of components of a sample based on charge status. In oneaspect of this embodiment, the CE is CZE. In another aspect of thisembodiment, the CE is CGE. In a further aspect of this embodiment, theCE is CLEF. In a final aspect of this embodiment, the CE is MEKC. In anyof these embodiments, the CE may be performed on a capillary arrayelectrophoresis instrument.

In one embodiment, when separation of the ionized components is doneusing IM, the present disclosure relates to use of drift time ionmobility spectrometry (DT-IMS), traveling wave ion mobility spectrometry(TW-IMS), or differential mobility spectrometry (DMS), which is alsoknown as fieldasymmetric ion-mobility spectrometry (FAIMS), for theseparation of ionized molecules of a sample according to the ionmobility of the ionized molecules. In one aspect of this embodiment, theionized molecules are separated according to ion mobility based on thecharge state of the ionized molecules.

In one embodiment, the present method includes the additional step ofionizing the components in the biological sample to be analyzed prior tothe separation of the ionized molecules of the sample according to theircharge state. In one aspect of this embodiment, the ionization step mustresult in ionization of at least two or more potential sites ofionization on the chemically tagged components in a sample. In oneaspect of this embodiment, the ionization may be achieved usingelectrospray ionization (ESI) or desorption electrospray ionization(DESI). In one aspect of this embodiment, the ionization step includeselectrospray ionization. In one embodiment, the ionization step occursafter the selective reaction of a component in a sample with a chemicaltag which has two or more sites for ionization.

Identification of Sample Components

In one embodiment, the present method includes identification of thecomponents of the sample using an analytical technique such as UVspectroscopy, flame ion detection, Raman spectroscopy, nuclearquadropole resonance (NQR), or mass spectrometry (MS). In anotherembodiment, the sample is analyzed to determine the identities of thecomponents therein using MS.

In one embodiment, the present method includes the separation of thecomponents in the sample is done using ion-mobility spectrometry (IM)and the analysis of the components of the sample is done using MS.

Mass Spectrometry (MS)

Mass spectrometry (MS) is an analytical technique that measures themass-to-charge ratio of a charged molecule or molecule fragments formedfrom a sample. MS is used to analyze the mass, chemical composition,and/or chemical structure of a component in a sample of interest. Ingeneral, MS includes three steps: ionizing a sample to form chargedmolecules or molecule fragments (i.e., ions); separating the ions basedon their mass-to-charge ratio; and detecting the separated ions to forma mass-to-charge signal (i.e., spectra).

A variety of mass spectrometry systems capable of high mass accuracy,high sensitivity, and high resolution are known in the art and can beemployed in the methods of the invention. The mass analyzers of suchmass spectrometers include, but are not limited to, quadrupole (Q), timeof flight (TOF), ion trap, magnetic sector or FT-ICR or combinationsthereof. The ion source of the mass spectrometer should yield mainlysample molecular ions, or pseudo-molecular ions, and certaincharacterizable fragment ions. Examples of such ion sources includeatmospheric pressure ionization sources, e.g. electrospray ionization(ESI) and Matrix Assisted Laser Desorption Ionization (MALDI). ESI andMALDI are the two most commonly employed methods to ionize proteins formass spectrometric analysis. ESI and APC1 are the most commonly used ionsource techniques for LC/MS (Lee, M. “LC/MS Applications in DrugDevelopment” (2002) J. Wiley & Sons, New York).

Various desorption ionization techniques have been combined IM and MSfor imaging of complex molecules, such as lipids, includingmatrix-assisted laser desorption ionization (MALDI), desorptionelectrospray ionization (DESI), rapid evaporative ionization MS, andlaser ablation electrospray ionization (LAESI). Any one of thesetechnique may be used in the present method, when analysis andidentification of the components of the sample is done using massspectrometry (MS). Any one of these techniques may be used in thepresent method, when analysis and identification of the components ofthe sample is done using mass spectrometry (MS). In one embodiment, thepresent disclosure includes the additional step of ionizing themolecules in the biological sample to be analyzed prior to theseparation of the ionized molecules of the sample according to theircharge state, wherein the ionization step is done using desorptionelectrospray ionization or laser ablation electrospray ionization. Inone embodiment, the method is performed using LAESI.

In one embodiment, the method is performed using LAESI. In anotherembodiment, the method is performed using DESI. In yet anotherembodiment, the method is performed using rapid evaporative ionizationMS. In still another embodiment, the method is not performed usingMALDI.

Commercially available mass spectrometers can sample and record thewhole mass spectrum simultaneously and with a frequency that allowsenough spectra to be acquired for a plurality of constituents in themixture to ensure that the mass spectrometric signal intensity or peakarea is quantitatively representative. This will also ensure that theelution times observed for all the masses would not be modified ordistorted by the mass analyzer and it would help ensure thatquantitative measurements are not compromised by the need to measureabundances of transient signals.

In another embodiment, the present disclosure includes the additionalstep of ionizing the molecules in the biological sample to be analyzedprior to the separation of the ionized molecules of the sample accordingto their charge state, wherein the ionization step is done usingdesorption electrospray ionization or laser ablation electrosprayionization.

Collision Cross Section (CCS)

Collision cross section (CCS) values are derived from ion mobilitymeasurements. All of the first order equations governing ion mobilityapply at low electric fields. Uniform field drift tube designs typicallyoperate at low electric field resulting in very predictable and accuratemobility measurements. Conventional uniform field drift tube ionmobility provides a direct method to calculate collision cross sections(W) using the Mason-Schamp equation given below:

$\Omega = {\frac{\left( {18\pi} \right)^{1/2}}{16}{\frac{ze}{\left( {k_{b}T} \right)^{1/2}}\left\lbrack {\frac{1}{m_{I}} + \frac{1}{m_{B}}} \right\rbrack}^{1/2}\frac{t_{d}E}{L}\frac{760}{P}\frac{T}{27{3.2}}\frac{1}{N}}$

where Ω is the rotationally averaged collision cross section, k_(b) isthe Boltzman constant, T is the temperature of the buffer gas, m_(l) isthe mass of analyte ion, m_(B) is the mass of buffer gas molecules,t_(d) is the corrected drift time, ze is the charge state of the analyteion, E is the electric field, L is the length of the drift cell, P isthe pressure in drift cell, and N is the number density in the driftcell. It is important to note that t_(d) can be determined from thetotal ion drift time. Once t_(d) values are calculated they can be usedto directly generate CCS measurements.

The accuracy to which the collision cross section can be calculated isdetermined by the extent to which experimental parameters (pressure,temperature and electric field) are maintained during the mobilityexperiment. Any time the ion spends outside of the defined drift regionproduces “end effects,” which cause loss of measurement accuracy.Measurements of CCS within 2% accuracy or less can be routinely achievedusing uniform field drift tubes. In one embodiment, the presentdisclosure relates to calculating a collisional cross section (CCS)value for the ionized components, wherein the CCS value assigned foreach molecule assists in the identification of the components of thesample. In addition to accurate mass, the experimental CCS of eachdetected component ion can be searched against CCS databases, to supportidentification. In one embodiment, components with chemical tags mayhave their CCS values selectively increased by reacting them withchemical tags of selected weight. In one aspect of this embodiment, CCSvalues are selectively increased by reacting components with chemicaltags of increasing size and weight. Identification and characterizationof the components of the sample may thus be enhanced using the presentmethod.

UV Spectroscopy

UV spectroscopy refers to absorption spectroscopy or reflectancespectroscopy in the ultraviolet-visible region. It employs light in thevisible, near-UV and near-infrared ranges to excite the components beingexposed to the light. The absorption or reflectance in the visible rangedirectly affects the perceived color of the chemicals involved. Thistechnique is analogous to fluorescence spectroscopy, except thatfluorescence deals with transitions from the excited state to the groundstate, and absorption measures transitions from the ground state to theexcited state. In one embodiment of the present method, the sample isanalyzed using UV spectroscopy after the separation by ion mobilityspectroscopy or capillary electrophoresis to identify and possiblyquantify the individual components of the sample.

Flame Ionization Detection (FID)

A flame ionization detector is an analytical instrument that measuresthe concentration of organic species in a gas stream. It is frequentlyused as a detector in gas chromatography. In one embodiment of thepresent method, the sample is analyzed using flame ion detection afterthe separation by ion mobility spectroscopy or capillary electrophoresisto identify and possibly quantify the individual components of thesample.

Raman Spectroscopy

Raman spectroscopy is a spectroscopic technique used to identifymolecules by their characteristic vibrational, rotational and otherlow-frequency modes. Raman spectroscopy as applies to the presentmethod, includes surface-enhanced Raman, resonance Raman, tip-enhancedRaman, polarized Raman, stimulated Raman, transmission Raman, spatiallyoffset Raman, and hyper Raman. In one embodiment of the present method,the sample is analyzed using Raman spectroscopy after the separation byion mobility spectroscopy or capillary electrophoresis to identify andpossibly quantify the individual components of the sample.

Nuclear Quadropole Resonance (NQR)

Nuclear quadropole resonance (NQR) is an analytic technique similar toNMR, except that the nuclei are excited by the interaction of anelectric field gradient and the quadropole moment of the nuclear chargedistribution. Each molecule has a unique NQR signal, and thus the methodcan be employed to specifically identify components of a sample. In oneembodiment of the present method, the sample is analyzed using nuclearquadropole resonance after the separation by ion mobility spectroscopyor capillary electrophoresis to identify and possibly quantify theindividual components of the sample.

Biological Samples

Biological samples can include any sample that is derived from the bodyof a subject. In this context, the subject can be an animal, for examplea mammal, for example a human. Other exemplary subjects include a mouse,rat, guinea-pig, rabbit, cat, dog, goat, sheep, pig, cow, or horse. Theindividual can be a patient, for example, an individual suffering from adisease or being suspected of suffering from a disease. A biologicalsample can be a bodily fluid or tissue, for example taken for thepurpose of a scientific or medical test, such as for studying ordiagnosing a disease (e.g., by detecting and/or identifying a pathogenor the presence of a biomarker). Biological samples can also includecells, for example, pathogens or cells of the individual biologicalsample (e.g., tumor cells). Such biological samples can be obtained byknown methods including tissue biopsy (e.g., punch biopsy) and by takingblood, bronchial aspirate, sputum, urine, feces, or other body fluids.Exemplary biological samples include humor, whole blood, plasma, serum,umbilical cord blood (in particular, blood obtained by percutaneousumbilical cord blood sampling (PUBS)), cerebrospinal fluid (CSF),saliva, amniotic fluid, breast milk, secretion, ichor, urine, feces,meconium, skin, nail, hair, umbilicus, gastric contents, placenta, bonemarrow, peripheral blood lymphocytes (PBL), and solid organ tissueextract.

In one embodiment, the sample is a blood sample, such as a dried bloodspot. In another embodiment, the sample is a blood-derived sample, suchas plasma or serum.

In another embodiment, the sample is a cell sample. The cell sample cancontain material obtained or derived from a subject. In otherembodiments, the cell sample can contain cells from an in vitro or exvivo cell culture. In other embodiments, the sample is a cellsupernatant sample.

EXAMPLES Example 1

Ionization sources based on electropspray (ESI), including DESI andLAESI, yields multiply charged ions. Charge separation provides that thecomponents in the sample are isolated separate from all other similarspecies based on charge state. FIG. 1 shows how charge state can beeffectively utilized to separate specific lipids out of a complex samplebased on charge state alone.

To exploit the use of CCS information of multiple charged ions toimprove MS-imaging applications, we analyzed human brain samples usingLAESI coupled to a IM-MS instrument. (See FIG. 1). Using CCS informationallowed for the isolation of lipids from metabolites, multiply chargedproteins and peptides, and from the background ions associated withatmospheric ionization. Identities of 93 lipid species were confirmedusing a combination of mass and CCS measurements. (FIG. 1, top).

Topographical maps representing the lipid ion distribution insub-regions of the human brain were created for selected mass and CCSvalues present in grey matter and white matter. The use of ion mobilityallowed the spatial separation of isobaric lipid species with differentCCS values, improving the quality of the signal-to-noise ratio. Notably,only by using this approach was it possible to determine the selectivespatial localization of gangliosides in the grey matter versus whitematter. These results indicate that CCS information of multiple chargedions may be a significant tool supporting lipid identification andlocalization in MS imaging studies. See FIG. 1 bottom, which clearlyshow that use of the IM-MS methodology of the present disclosure leadsto greatly enhanced accuracy and precision for measuring the spatiallocalization of components in tissue cross sections.

Example 2

FIG. 2 shows a direct analysis of gangliosides from dried blood spots(DBS) by DESI. Imaging was done using a SYNAPT G2-Si HDMS (WATERS®)equipped with a 2D-DESI source. FIG. 2 also shows that multiply chargedions are clearly separated from the sample using the combined techniqueIM-MS, whereas characterization of individual components was not at alleffective without ion mobility separation.

Data was generated and analyzed using WATERS® High Definition ImagingSoftware (HDI) 1.35. Spray conditions were as follows: flow rate of 1μL/min, with a 98% methanol in water mixture at 100 psi N₂ gas pressureand a voltage of 5 kV for both polarities. The scan time was 1 second.Gangliosides and cardiolipins (not shown) were separated out as doublycharged ion species, which allowed them to be isolated using ionmobility against interfering ions.

1. A method of identifying components of a sample comprising: a)selectively reacting a component in the sample with a chemical tag whichhas two or more sites for ionization; b) separating the components usingion mobility spectrometry or capillary electrophoresis; and c) analyzingthe sample to identify the components therein.
 2. The method of claim 1,wherein the separation according to ion mobility spectrometry separatescomponents based on the charge state of the component molecules. 3-6.(canceled)
 7. The method of claim 1, wherein the sample is analyzedusing an electrophoretic technique. 8-10. (canceled)
 11. The method ofclaim 1, wherein the separation of the components in step b) is doneusing ion-mobility spectrometry (IM) and the analysis of the sample instep c) is done using MS. 12-13. (canceled)
 14. The method of claim 1,wherein the chemical tag contains a moiety which absorbs in the visible(VIS) spectrum or the infrared (IR) spectrum. 15-27. (canceled)
 28. Themethod of claim 1, further comprising calculating a collisional crosssection (CCS) value of the ionized molecules, wherein the collisioncross section value assists in the identification of components in thesample.
 29. (canceled)
 30. The method of claim 1, wherein the chemicaltag comprises two or more functional groups which are readily ionizable,each independently selected from amino, carboxylic acid, ester,carbamate and/or phosphonate. 31-33. (canceled)
 34. The method of claim30, wherein the chemical tag is a dendrimer-type molecule containing twoor more ionizable groups.
 35. The method of claim 34, wherein thedendrimer-type molecule is polyamidoamine (PAMAM), polypropyleneimine(PPI), polyglutamic acid and/or polyester dendrimer.
 36. The method ofclaim 30, wherein the chemical tag is selected from a compound accordingto formula (I), (II) and (III):

or a salt thereof, wherein: each Y is independently —(CH₂CH₂)_(n)W—,—(CH₂)_(n)W—, (CH₂CH₂W)_(n), —(CH₂W)_(n)(CH₂)_(k)—,—(CH₂CH₂W)_(n)(CH₂)_(k)—, —(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—,

each Y′ is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(k)—,—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—,

each Y″ is independently —(CH₂CH₂)_(n)W—, —(CH₂)_(n)W—, (CH₂CH₂W)_(n),—(CH₂W)_(n)(CH₂)_(k)—, —(CH₂CH₂W)_(n)(CH₂)_(k)—, or—(CH₂)_(p)—W—C(O)—W—(CH₂)_(p)—; each Y₁ is independently:—(CH₂)_(n)W(CH₂)_(n)—, —OC(O)—(CH₂)₁₋₄—, —OC(O)—(CH₂)₁₋₄W— or absent;each X is independently: —N⁺(C₁-C₆alkyl)₃, —S(O)₂(OH), —OS(O)₂(OH),—NH—C(═NH)NH₂, —OP(O)(OH)₂, —P(O)(OH)₂, —C(O)N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)₂, —NHC(O)(OC₁-C₆alkyl), —CO₂H, —C(O)NH₂,—C(O)NH(C₁-C₆alkyl), or C(O)(OC₁-C₆alkyl); each RG is independentlyisocyanate, thioisocyanate, succinimidyl ester, succinimidyl carbamate,carboxylic acid, amine, or aldehyde; each W is independently N(C₁-C₆alkyl) NH, O, S, or absent; each Z is independently CH or N; each R isindependently halo, —OH, —CN, C₁-C₃ alkyl, C₃-C₆ cycloalkyl, C₁-C₃alkoxy, C₁-C₃ haloalkyl or C₁-C₃ haloalkoxy; each n is independently0-4; each p is independently 0-6; each k is independently 0-6; and m is0-3; and wherein each (CH₂) or (CH₂CH₂) segment of Y, Y′ and Y″ may beoptionally and independently substituted with 1 or 2 groups selectedfrom methyl, ethyl, —OH, halo or carbonyl and wherein each Y, Y′ and Y″is attached to an X group; provided that n and k are not both 0 when Y,Y′ or Y″ is —(CH₂W)_(n)(CH₂)_(k)— or —(CH₂CH₂W)_(n)(CH₂)_(k)—.
 37. Themethod of claim 36, wherein the chemical tag is according to formula(Ia):

or a salt thereof.
 38. The method of claim 36, wherein the chemical tagis according to formula (IIa):

or a salt thereof.
 39. The method of claim 36, wherein the chemical tagis according to formula (IIIa):

or a salt thereof.
 40. The method of claim 37, wherein the chemical tagis according to formula (Ib):

or a salt thereof.
 41. The method of claim 38, wherein the chemical tagis according to formula (IIb):

or a salt thereof.
 42. The method of claim 39, wherein the chemical tagis according to formula (IIIb):

or a salt thereof. 43-44. (canceled)
 45. The method of claim 36, whereinthe chemical tag is selected from one of the formulae:

or a salt thereof; wherein each Q, if present, is N(C₁-C₃alkyl), NH orO; each T, if present, is H, methyl or OH; each Y₁ is —(CH₂)₁₋₃ NH—,—OC(O)(CH₂)₁₋₃—, —NHC(O)(CH₂)₁₋₃—, —C(O)O(CH₂)₁₋₃—, —C(O)NH(CH₂)₁₋₃—,—(CH₂)₁₋₃—, or absent; each X is independently —N⁺(C₁-C₆alkyl)₃,—S(O)₂(OH), —OS(O)₂(OH), —NHC(═NH)NH₂, —OP(O)(OH)₂, P(O)(OH)₂,—N(C₁-C₆alkyl)₂, or —CO₂H; and each RG is isocyanate, thioisocyanate,succinimidyl ester or a succinimidyl carbamate. 46-49. (canceled) 50.The method of claim 30, wherein the chemical tag is selected from thegroup consisting of:

or a salt thereof.
 51. A compound selected from:

or a salt thereof; wherein each R³ is independently C₁-C₆ alkyl; andeach R⁴ is independently —H or C₁-C₆ alkyl.
 52. (canceled)
 53. Themethod of claim 30, wherein the chemical tag is a compound selectedfrom:

or a salt thereof; wherein each R³ is independently C₁-C₆ alkyl; andeach R⁴ is independently —H or C₁-C₆ alkyl.